The inducible deletion of Drosha and microRNAs in mature podocytes results in a collapsing glomerulopathy

Micro-RNAs (miRNAs) are short (average 22 nucleotides) noncoding regulatory RNAs that inhibit gene expression by targeting complementary 3'-untranslated regions of protein-encoding mRNAs for translational repression or degradation. miRNAs play key roles in both the function and differentiation of many cell types. Drosha and Dicer, two RNAase III enzymes, function in a stepwise manner to generate a mature miRNA. Previous studies have shown that podocyte-specific deletion of Dicer during development results in proteinuric renal disease and collapsing glomerulopathy (CG); however, Dicer has functions other than the generation of miRNAs. Here we found that the podocyte-specific deletion of Drosha results in a similar phenotype to Dicer mutants, confirming that the Dicer mutant phenotype is due to the loss of miRNAs. Moreover, the inducible deletion of Drosha in 2- to 3-month-old mice (Tet-On system) resulted in CG. Thus, continuous generation of miRNAs are required for the normal function of mature podocytes and their loss leads to CG. Identifying these miRNAs may provide new insight into disease pathogenesis and novel therapeutic targets in various podocytopathies.     

The next level of complexity: post-transcriptional regulation by microRNAs

MicroRNAs (miRNAs) are endogenous, small, noncoding RNAs that regulate expression of about half of human genes. Canonical miRNA biosynthesis involves two RNase III-type proteins, Drosha and Dicer. A podocyte-specific knockout of Dicer resulted in foot process effacement. In this issue, Zhdanova et al. report on targeting Drosha in a podocyte-specific fashion. Their study demonstrates an essential role of canonical miRNAs in maintaining the fully differentiated podocyte phenotype.     

Expression of sulfotransferase 1E1 in human prostate as studied by in situ hybridization and immunocytochemistry

BACKGROUND: Estrogen is recognized to play a role in the development and function of the prostate. Estrogen sulfotransferase (EST) 1E1 catalyzes the sulfoconjugation of estrogen and is thus involved in the metabolism of estrogen. We have recently shown that EST 1E1 is highly expressed in male mouse reproductive organs, including prostate. It appeared of interest to study the expression of EST 1E1 in human prostate. METHODS: EST 1E1 mRNA and protein expression was evaluated in benign prostatic hyperplasia (BPH) using in situ hybridization and immunocytochemistry, respectively. RESULTS: EST 1E1 mRNA and protein were found to be expressed in epithelial cells bordering alveola lumen (luminal cells) as well as stroma cells. CONCLUSION: The enzyme EST may play a physiological role in regulating local estrogen levels in human prostate.     

Review: The role of microRNAs in kidney disease

MicroRNAs (miRNAs) are short non-coding RNAs that modulate physiological and pathological processes by inhibiting target gene expression via blockade of protein translation or by inducing mRNA degradation. These miRNAs potentially regulate the expression of thousands of proteins. As a result, miRNAs have emerged rapidly as a major new area of biomedical research with relevance to kidney disease. MiRNA expression has been shown to differ between the kidney and other organs as well as between different kidney regions. Furthermore, miRNAs have been found to be functionally important in models of podocyte development, diabetic nephropathy and polycystic kidney disease. Of particular interest, podocyte-specific deletion of Dicer, a key enzyme in the biogenesis of miRNA, results in proteinuria and severe renal impairment in mice. One miRNA (miR-192) can also act as an effector of transforming growth factor-β activity in the high-glucose environment of diabetic nephropathy. Differential expression of miRNAs has been reported in kidney allograft rejection. It is anticipated that future studies involving miRNAs will generate new insights into the complex pathophysiology underlying various kidney diseases, generate diagnostic biomarkers and might be of value as therapeutic targets for progressive kidney diseases. The purpose of this review is to highlight key miRNA developments in kidney diseases and how this might influence the diagnosis and management of patients with kidney disease in the future.     

Localization of androgen and estrogen receptors in adult male mouse reproductive tract

There is considerable variation, both within and between species, in reports of nuclear steroid receptor localizations in the male reproductive tract. In this study, androgen receptor (AR) and estrogen receptors ERalpha and ERbeta were visualized by immunohistochemistry in adult male mice reproductive tracts, including testes, efferent ductules; initial segment, caput, corpus, and cauda epididymides; and vas deferens. Antibody specificity was demonstrated by Western blot and antibody competition. In testis, AR was expressed in Leydig cells, Sertoli cells, and most peritubular cells, but not in germ cells; Sertoli cells showed more intense staining in stages VI-VII; ERalpha was present in Leydig and some peritubular cells; ERbeta was in Leydig, some peritubular, all Sertoli and germ cells except in spermatids and meiotic spermatocytes. In efferent ductules, AR was strongly expressed in ciliated and nonciliated epithelial cells and in stromal cells; ERalpha was strongly expressed in ciliated and nonciliated epithelial cells; stromal cells were negative; and ERbeta was strongly expressed in ciliated and nonciliated epithelial cells and also in stromal cells. In epididymis, AR was strongly expressed in all epithelial cells (not in intraepithelial lymphocytes); ERalpha was strongly expressed in apical, narrow, and some basal cells of the initial segment, and in caput, principal cells of the caput, clear cells of the distal caput through cauda; stromal cells were negative in the initial segment, but more stromal cells were stained from caput to cauda; ERbeta was strongly expressed in most of epithelial cells of the epididymis, but stromal cells were inconsistently stained. In vas deferens, AR was weakly expressed or absent in principal cells but moderately stained in basal cells, smooth muscle cells of stroma were stained intensely, ERalpha was absent in epithelial cells but present in a subepithelial smooth muscle layer, and ERbeta was strongly expressed in all epithelial cells and most stromal cells. This study demonstrates that the reproductive tracts of male mice differ considerably from those of rats in expression of ARs and ERs and that caution is needed when extrapolating nuclear steroid receptor data across mammalian species.     

Survivin对PC-3细胞微小RNA表达的影响

目的 检测Survivin抑制后PC-3细胞微小RNA( miRNAs)表达谱的变化,探讨Survivin与相关miRNAs之间的关系.方法 利用负载Survivin短发夹RNA (shRNA)重组腺病毒( pGSadeno-sur shRNA),体外转染PC-3细胞,96 h后,收集细胞提取RNA,检测miRNA表达谱的变化.结果 共有650个miRNAs表达信号在雄激素非依赖前列腺癌PC-3细胞中可以被检测到,有9个miRNAs的表达信号水平高于1000,其中的6个miRNAs表达谱同实验组比较差异有统计学意义(P＜0.01).实验组与空白对照组比较有75个miRNAs表达差异有统计学意义(P＜0.01),其中42个表达上调、33个表达下调;阴性对照组与实验组比较有27个miRNAs表达差异有统计学意义(P＜0.01),其中22个表达上调、5个表达下调;实验组与空白对照组及阴性对照组比较均表达上调的miRNAs有15个,表达下调的miRNA有1个.结论 抑制PC-3细胞中Survivin的表达对miRNAs 表达谱有明显影响,为明确相关miRNAs在PC-3细胞中的作用提供新线索.     

Cholinergic innervation and muscarinic receptors in the human prostate

OBJECTIVE: In light of recent interest in the use of muscarinic receptor antagonists for the treatment of male lower urinary tract symptoms, understanding how such drugs work not only on the bladder but also on the prostate is important. METHODS: A literature review was conducted to identify studies on the cholinergic innervation and presence and function of muscarinic acetylcholine receptors in the human prostate. RESULTS: The available studies demonstrate a dense cholinergic innervation within both stromal and epithelial compartments of the prostate. Concomitantly, the human prostate expresses muscarinic receptors at densities exceeding those of alpha(1)-adrenoceptors. They mainly belong to the M(1) subtype and are found on epithelial cells, but a smaller population of M(2) receptors is found on stromal cells. Both populations have been shown to be functional in signal transduction assays. However, in line with the sparse receptor density on stromal smooth muscle cells, contractile responses of the prostate are only small. Data from prostate cancer cell lines and from botulinum toxin injections into the benign prostate raise the possibility that muscarinic receptors may promote prostatic growth. Animal data suggest that muscarinic receptors may be of primary importance in the genesis of prostatic secretions, but this needs to be confirmed in humans. CONCLUSIONS: Taken together it appears that direct effects on the prostate need to be considered when using muscarinic receptor antagonists in men. They may primarily involve alterations of glandular secretion and prostatic growth.     

Prosaposin ablation inactivates the MAPK and Akt signaling pathways and interferes with the development of the prostate gland

The recent development of a prosaposin-/-mouse model has allowed the investigation of the role of prosaposin in the development of the male reproductive organs.A morphometric analysis of the male reproductive system of 37 days old mice revealed that prosaposin ablation produced a 30% reduction in size and weight of the testes,37% of the epididymis,75% of the seminal vesicles and 60% of the prostate glands.Light microscopy(LM) showed that smaller testis size from homozygous mutant mice was associated with reduced spermiogenesis.Both,dorsal and ventral lobules of the prostate glands were underdeveloped in the homozygous mutant.LM analysis also showed that prostatic alveoli were considerably smaller and lined by shorter epithelial cells in the homozygous mutant.Smaller tubular diameter and shorter undifferentiated epithelial cells were also observed in seminal vesicles and epididymis. In the efferent ducts of the homozygous mutant mice,the epithelium was composed exclusively of ciliated cells in contrast to the heterozygotes,which showed the presence of nonciliated cells.Radioimmunoassays demonstratedthat testosterone levels were normal or higher in mice with the inactivated prosaposin gene.Immunostaining of prostate sections with an anti-androgen receptor antibody showed that the epithelial cells lining the alveoli express androgen receptor in both the heterozygous and homozygous tissue.Similarly,sections immunostained with antibodies to the phosphorylated MAPKs and Akts strongly reacted with tall prostatic secretory cells in prostate from heterozygous mouse.On the other hand,the epithelial cells in the homozygous prostate remained unstained or weakly stained.These findings demonstrate that inactivation of the prosaposin gene affected the development of the prostate gland and some components of the MAP pathway.     

微小核糖核酸与前列腺癌

微小核糖核酸(microRNAs,miRNAs)是一类长度约为22个核苷酸,参与基因转录后水平调控的非编码小分子RNA。参与生命的多个过程且与癌症发生发展有着密切的关系。最近研究发现,一些miRNA在前列腺癌(PCa)组织中异常表达,表明miRNA在PCa发病的分子机制中起着重要作用。随着人血清/血浆miRNA的不断深入研究,血清miRNA有望作为诊断PCa的潜在分子标志物。     

Suppression of androgen action and the induction of gross abnormalities of the reproductive tract in male rats treated neonatally with diethylstilbestrol

This study evaluated whether androgen action is altered in rats treated neonatally with diethylstilbestrol (DES) at a dose that induced reproductive tract abnormalities. Rats were treated on alternate days 2-12 with 10 microg DES and studied on Day 18. DES-induced abnormalities included a 70% reduction in testis weight, distension and overgrowth of the rete, distension and reduction in epithelial height of the efferent ducts, underdevelopment of the epididymal duct epithelium, reduction in epithelial height in the vas deferens, and convolution of the extra-epididymal vas. In DES-treated rats, androgen receptor (AR) immunoexpression was virtually absent from all affected tissues and the testis, whereas AR expression in controls was intense in epithelial and stromal cells. The DES-induced change in AR immunoexpression was confirmed by Western analysis for the testis. In rats treated neonatally with 1 microg DES, reproductive abnormalities were absent or minor, except for a 38% reduction in testis weight; loss of AR immunoexpression also did not occur in these rats. Treatment-induced changes in AR expression were paralleled by changes in Leydig cell volume per testis (91% reduction in the 10-microg DES group; no change in the 1-microg DES group). To test whether suppression of androgen production or action alone could induce comparable reproductive abnormalities to 10 microg DES, rats were treated neonatally with either a potent gonadotropin-releasing hormone antagonist (GnRHa) or with flutamide (50 mg/kg/day). These treatments reduced testis weight (68% for GnRHa, 40% for flutamide), and generally retarded development of the reproductive tract but failed to induce the abnormalities induced by 10 microg DES. GnRHa and flutamide caused no detectable change in AR immunoexpression in target tissues, with the exception of minor changes in the testes of flutamide-treated males. GnRHa treatment caused a reduction (83%) in Leydig cell volume comparable to that caused by 10 microg DES. Immunoexpression of estrogen receptor alpha (ER alpha) in the efferent ducts and of ER beta in all tissues studied were unaffected by any of the above treatments. Neonatal coadministration of testosterone esters (TE; 200 microg) with 10 microg DES prevented most of the morphological abnormalities induced by 10 microg DES treatment alone, though testis weight was still subnormal (46% reduction in DES + TE vs 72% in DES alone and 49% with TE alone) and some lumenal distension was still evident in the efferent ducts. Coadministration of TE with DES prevented DES-induced loss of AR immunoexpression (confirmed for testis by Western blot analysis). It is concluded that 1) reproductive tract abnormalities induced in the neonatal male rat by a high (10 microg) dose of DES are associated with reduced AR expression and Leydig cell volume; 2) these changes are largely absent with a lower dose of DES (1 microg); 3) treatments that interfere with androgen production (GnRHa) or action (flutamide) alone failed to induce reproductive tract abnormalities or alter AR expression as did 10 microg DES; 4) a grossly altered androgen:estrogen balance (low androgen + high estrogen) may underlie the reproductive tract abnormalities, other than reduced testis weight, induced by high doses of DES.     

Prostanoid transport by multidrug resistance protein 4 (MRP4/ABCC4) localized in tissues of the human urogenital tract

PURPOSE: The seminal vesicles are the major source of prostaglandins in seminal fluid. For prostanoid action on cell surfaces they must be released from synthesizing cells. MRP4/ABCC4 (multidrug resistance protein 4 adenosine triphosphate-binding cassette, subfamily C, member 4) is an adenosine triphosphate dependent export pump for organic anions that may mediate prostanoid transport across the plasma membranes. Therefore, we analyzed whether MRP4 is expressed in the seminal vesicles and other tissues of the human urogenital tract, whether MRP4 and prostanoid synthesizing enzymes are co-expressed in the same cell type and whether MRP4 functions as a prostanoid export pump. MATERIALS AND METHODS: The expression and localization of MRP4 and prostanoid synthesizing enzymes were investigated in several tissues of the male human urogenital tract by immunoblot and immunofluorescence analyses. Prostanoid transport was measured into inside-out membrane vesicles from cells expressing recombinant human MRP4. RESULTS: MRP4 and prostanoid synthesizing enzymes were co-expressed in the epithelial cells of human seminal vesicles. Moreover, MRP4 was localized in the plasma membrane of epithelial cells of the ureter, in the basolateral membrane of glandular epithelial cells of the prostate, and in smooth muscle cells of the bladder and corpus cavernosum. Transport studies established MRP4 as an efflux pump for prostaglandin E2 (Michaelis constant [Km] 3.5 muM), thromboxane B2 (Km 9.9 muM) and prostaglandin F2alpha (Km 12.6 muM). CONCLUSIONS: The co-expression of prostanoid synthesizing enzymes and MRP4 in epithelial cells of the human seminal vesicles and the function of MRP4 as a prostanoid efflux pump indicate that MRP4 mediates prostanoid transport from these cells, which are the main prostanoid synthesizing cells in the male urogenital tract.